This application is filed pursuant to 35 USC xc2xa7371 based upon PCT/US98/1867, filed Sep. 8, 1998, which claims priority to GB 9719637.2 filed Sep. 15, 1997.
The present invention relates to metallo-proteases derived from gram-positive microorganisms. The present invention provides nucleic acid and amino acid sequences of metallo-proteases identified in Bacillus. The present invention also provides methods for the production of the metallo-protease in host cells as well as the production of heterologous proteins in a host cell having a mutation or deletion of part or all of metallo-proteases of the present invention.
Gram-positive microorganisms, such as members of the group Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media. In gram-positive bacteria, secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually maintaining their native conformation.
Various gram-positive microorganisms are known to secrete extracellular and/or intracellular protease at some stage in their life cycles. Many proteases are produced in large quantities for industrial purposes. A negative aspect of the presence of proteases in gram-positive organisms is their contribution to the overall degradation of secreted heterologous or foreign proteins.
The classification of proteases found in microorganisms is based on their catalytic mechanism which results in four groups: the serine proteases; metallo-proteases; cysteine proteases; and aspartic proteases. These categories can be distinguished by their sensitivity to various inhibitors. For example, the serine proteases are inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate (DIFP); the metallo-proteases by chelating agents; the cysteine enzymes by iodoacetamide and heavy metals and the aspartic proteases by pepstatin. The serine proteases have alkaline pH optima, the metalloproteases are optimally active around neutrality, and the cysteine and aspartic enzymes have acidic pH optima (Biotechnology Handbooks, Bacillus. vol. 2, edited by Harwood, 1989 Plenum Press, New York).
Metallo-proteases form the most diverse of the catalytic types of proteases. About half of the families comprise enzymes containing the His-Glu-Xaa-Xaa-His (or HEXXH) motif which has been shown by X-ray crystallography to form part of the site for binding of the metal (normally zinc) atom. In one family of metalloproteases, a glutamic acid residue completes the metal-binding site, HEXXH+E. This family contains the most well characterized of the metallo-proteases, thermolysin. The three dimensional structure of thermolysin shows that, in the HEXXH motif, the His residues are zinc ligands and the Glu residue has a catalytic function. (Methods in Enzymology, vol. 248, Academic Press, Inc. 1994).
Fujimura-Kamada et al. (1997, J. Cell Biol. 136: 271-285) disclose a new subfamily of proteins that appear to function as intracellular, membrane-associated zinc metalloproteases. They disclose the Saccharomyces cerevisiae STE24 gene product which contains a zinc metalloprotease motif (HEXXH), as well as multiple predicted membrane spans. They further disclose that STE24 is required for the first NH2-terminal proteolytic cleavage event during biogenesis of the a-factor precursor.
The present invention relates to the discovery of a heretofore unknown metallo-protease (MP) found in gram positive microorganisms, uses of the MP in industrial applications, and advantageous strain improvements based on genetically engineering such microorganisms to delete, underexpress or overexpress that MP. The present invention is based in part upon the discovery that MP has overall amino acid relatedness to S. cerevisiae STE24 (Fujimura-Kamada et al., supra) and in part upon the unexpected discovery that nucleic acid encoding gram positive microorganism MP is found immediately downstream of nucleic acid encoding the major alkaline protease putative transcriptional terminator in gram-positive microorganisms.
The present invention is also based, in part, upon Applicant""s discovery that the characteristic metallo-protease amino acid motif HEXXH+E and putative transmembrane domains exist in Bacillus subtilis MP. The present invention is also based in part upon Applicant""s discovery that Bacillus subtilis MP homologs are found in Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens. Applicant""s discovery, in addition to providing a new and useful group of proteases and methods of detecting DNA encoding such proteases in a gram positive microorganism, provides several advantages which may facilitate optimization and/or modification of strains of gram positive microorganisms, such as Bacillus, for expression of desired, e.g. heterologous, proteins. Such optimizations, as described below in detail, allow the construction of strains having decreased proteolytic degradation of desired expression products.
Due to the relatedness of MP to STE24, a zinc metallo-protease which has been shown to be involved in processing events, and the unexpected conserved structural arrangement and proximity of gram positive MPs to the major alkaline protease of multiple Bacillus species, it appears that MP may play a role in regulating and/or processing the major alkaline protease in Bacillus. Furthermore, MP can serve as a marker for identification of the major alkaline protease in Bacillus species.
In one embodiment, the metallo-protease is obtainable from a gram-positive microorganism which is a Bacillus. In another embodiment, the metallo-protease is obtainable from a Bacillus which is preferably selected from the group consisting of Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis and Bacilliis amyloliquifaciens. The present invention encompasses the naturally occurring MP encoded by nucleic acid found immediately downstream from the transcriptional terminator of the major alkaline protease of a Bacillus species as well as the nucleic acid and amino acid molecules having the sequences disclosed in the Figures.
In a preferred embodiment, the present invention encompasses the naturally occurring MP nucleic acid molecule having the sequence found in Bacillus subtilis I-168 strain (Bacillus Genetic Stock Center, accession number 1A1, Columbus, Ohio) in the region of about 1102 kb from the point of origin and immediately downstream of the putative transcriptional terminator of the aprE gene. In another preferred embodiment, the Bacillus subtilis MP nucleic acid and amino acid molecules have the sequences as shown in FIGS. 1A-1E.
The present invention is also based in part upon the unexpected discovery of nucleic acid encoding portions of Bacillus subtilis MP homologs found in at least 3 non B. subtilis Bacillus species, including Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens. The Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens MP is found downstream of the major alkaline protease of each Bacillus.
The present invention encompasses the naturally occurring Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens MP. In a preferred embodiment, the MP is encoded by the nucleic acid molecules having the nucleic acid sequence that is immediately downstream of the putative transcriptional terminator of the major alkaline protease or subtilisn in the genome of Bacillus stearothermophilus, Bacillus licheniformis or Bacillus amyloliquifaciens. 
In one preferred embodiment, the Bacillus stearothermophilus MP comprises the amino acid sequence as shown in FIG. 3. In another preferred embodiment, the Bacillus licheniformis MP comprises the amino acid sequence as shown in FIG. 4. In another preferred embodiment, the Bacillus amyloliquifaciens MP comprises the amino acid sequence as shown in FIG. 5. The present invention encompasses any nucleic acid molecule encoding Bacillus stearothermophilus, Bacillus lichenifonnis or Bacillus amyloliquifaciens MP.
The present invention provides isolated polynucleotide and amino acid sequences for Bacillus subtilis MP in FIGS. 1A-1E. Due to the degeneracy of the genetic code, the present invention encompasses any nucleic acid sequence that encodes the Bacillus subtilis MP amino acid sequence. The present invention provides expression vectors and host cells comprising nucleic acid encoding a gram-positive MP. The present invention also provides methods of making the gram positive MP.
The present invention encompasses novel amino acid variations of gram positive MP amino acid sequences disclosed herein that have proteolytic activity. Naturally occurring gram positive MP as well as proteolytically active amino acid variations or derivatives thereof, have application in the textile industry, in cleaning compositions and in animal feed.
The present invention provides methods for detecting gram positive microorganism homologs of B. subtilis MP that comprises hybridizing part or all of the nucleic acid encoding B. subtilis MP with nucleic acid derived from gram-positive organisms, either of genomic or cDNA origin. Accordingly, the present invention provides a method for detecting a gram-positive microorganism MP, comprising the steps of hybridizing gram-positive microorganism nucleic acid under low stringency conditions to a probe, wherein the probe comprises part or all of the nucleic acid sequence shown in FIGS. 1A-1E; and isolating gram-positive nucleic acid which hybridizes to said probe.
Based upon the genomic proximity of MP to the major alkaline protease, the present invention provides a means of detecting the major alkaline protease of gram-positive microorganisms species based upon nucleic acid hybridization to B. subtilis MP. In one embodiment, the gram-positive microorganism is a Bacillus. In another preferred embodiment, the Bacillus is selected from the group consisting of B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis. 
The production of desired heterologous proteins or polypeptides in gram-positive microorganisms may be hindered by the presence of one or more proteases which degrade the produced heterologous protein or polypeptide. One advantage of the present invention is that it provides methods and expression systems which can be used to prevent that degradation, thereby enhancing yields of the desired heterologous protein or polypeptide.
Accordingly, the present invention provides a gram-positive microorganism having a mutation or deletion of part or all of the gene encoding MP, which results in the inactivation of the MP proteolytic activity, either alone or in combination with mutations in other proteases, such as apr, npr, epr, mpr, bpf or isp for example, or other proteases known to those of skill in the art. In one embodiment of the present invention, the gram-positive organism is a member of the genus Bacillus. In another embodiment, the Bacillus is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulates, B. circulans, B. lautus and Bacillus thuringiensis. In a further preferred embodiment, the Bacillus is Bacillus subtilis. 
The present invention also encompasses amino acid variations or derivatives of gram positive MP that do not have the characteristic proteolytic activity as long as the nucleic acid sequences encoding such variations or derivatives would have sufficient 5xe2x80x2 and 3xe2x80x2 coding regions to be capable of being integrated into a gram-positive organism genome. Such variants would have applications in gram-positive expression systems where it is desirable to delete, mutate, alter or otherwise incapacitate the naturally occurring metallo-protease in order to diminish or delete its proteolytic activity. Such an expression system would have the advantage of allowing for greater yields of recombinant heterologous proteins or polypeptides.
In another aspect, the gram-positive host having one or more metallo-protease deletions or mutations is further genetically engineered to produce a desired protein. In one embodiment of the present invention, the desired protein is heterologous to the gram-positive host cell. In another embodiment, the desired protein is homologous to the host cell. The present invention encompasses a gram-positive host cell having a deletion, mutation or interruption of the nucleic acid encoding the naturally occurring homologous protein, such as a protease, and having nucleic acid encoding the homologous protein re-introduced in a recombinant form. In another embodiment, the host cell produces the homologous protein. Accordingly, the present invention also provides methods and expression systems for reducing degradation of heterologous proteins produced in gram-positive microorganisms. The gram-positive microorganism may be normally sporulating or non-sporulating. In a preferred embodiment, the gram positive host cell is a Bacillus. In another preferred embodiment, the Bacillus host cell is Bacillus. In another embodiment, the Bacillus is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis. 
Naturally occurring gram positive MP as well as proteolytically active amino acid variations or derivatives thereof, have application in the textile industry, in cleaning compositions and in animal feed. The metallo-protease MP may be used alone or in combination with other enzymes and/or mediators or enhancers. Accordingly, the present invention provides a cleaning composition comprising a metalloprotease of the present invention having the amino acid sequence shown in FIGS. 1A-1E or the amino acid encoded by the MP nucleic acid found at about 1102 kilobases from the point of origin of Bacillus subtilis. Also provided are cleaning compositions comprising a metalloprotease having at least 80%, at least 90%, or at least 95% homology with the amino acid sequence shown in FIGS. 1A-1E or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in FIGS. 1A-1E under high stringency conditions.
Further there is provided an animal feed comprising a metalloprotease, MP, having the amino acid sequence shown in FIGS. 1A-1E. Also provided are animal feeds comprising a metalloprotease having at least 80%, at least 90%, and at least 95% homology with the amino acid sequence shown in FIGS. 1A-1E or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in FIGS. 1A-1E under high stringency conditions.
Also provided is a composition for the treatment of a textile comprising a metalloprotease, MP, having the amino acid sequence shown in FIGS. 1A-1E. Also provided are compositions for the treatment of a textile comprising a metalloprotease having at least 80%, at least 90%, or at least 95% homology with the amino acid sequence shown in FIGS. 1A-1E or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in FIGS. 1A-1E under high stingency conditions.
In a further aspect of the present invention, a Gram-positive MP is produced on an industrial fermentation scale in a microbial host expression system.